Ancient proteins resolve controversy over the identity ofGenyorniseggshell

Abstract

Significance The controversy over the taxonomic identity of the eggs exploited by Australia’s first people around 50,000 y ago is resolved. The birds that laid these eggs are extinct, and distinguishing between two main candidates, a giant flightless “mihirung” Genyornis and a large megapode Progura , had proven impossible using morphological and geochemical methods. Ancient DNA sequencing remains inconclusive because of the age and burial temperature of the eggshell. In contrast, ancient protein sequences recovered from the eggshell enabled estimation of the evolutionary affinity between the egg and a range of extant taxa. The eggs are those of a Galloanseres (a group that includes extinct Dromornithidae, as well as extant landfowl and waterfowl), Genyornis , and not of the megapode (Megapodiidae, crown Galliformes).

Survival of mineral-bound peptides into the Miocene

“…The sequence recovered from the Chinese specimen is identical to the peptides found in the 3.8 Ma Laetoli OES, thus also supporting the attribution of the Liushu ootaxon to genus Struthio. While the four Asp residues are conserved across several avian taxa, in some species, including other ratites, Asp can be substituted by Glu and some of the flanking residues are also variable (Demarchi et al, 2022, fig. S1); therefore, variability within this sequence could be informative for evolutionary relationships between extinct and extant taxa.…”

“…A subsample of OES specimen IVPP V26107 was prepared in the ultra-clean facility at the University of Copenhagen, following the protocol of Demarchi et al (2022Demarchi et al ( , 2016 and omitting the digestion step. In brief, the fragment was powdered, bleached for 72 hours (NaOCl, 15% w/v) and demineralised in just enough cold weak hydrochloric acid (0.6 M HCl).…”

Molecular exploration of fossil eggshell uncovers hidden lineage of giant extinct bird

“…While proteins typically provide less phylogenetic signal than ancient DNA, we nevertheless sequenced the ancient proteins preserved in the eggshell matrix to explore amino acid differences. Partial sequences of putative nuclear-encoded type-1 and type-2 Clectin eggshell proteins 21 (XCA-1, XCA-2) reconstructed from 12 eggshell specimens (Supplementary Data 7) support a family-level distinction between mullerornithids and aepyornithids, with all putatively mullerornithid eggshell (<1.5 mm thickness) having a histidine at residue 74 of the XCA-1 alignment, and all aepyornithid eggshell (>1.5 mm thickness) having a tyrosine at this site (Supplementary Figs. 4-5).…”

“…Deamidation of N and Q, as well as oxidation of M, H, and W were set as variable PTMs. The files were searched against all available XCA-1 and XCA-2 protein sequences 21 , and the common Repository of Adventitious Proteins (cRAP) to identify common contaminants. The proteomics datasets have been deposited to the Pro-teomeXchange Consortium via the Proteomics Identifications Database (PRIDE) partner repository with the dataset identifier PXD035725.…”

Current developments and future directions in archaeological science

“…Demarchi et al. ( 53 ) apply a paleoproteomic approach to identify the avian taxon responsible for Late Pleistocene eggs that were exploited by Indigenous people in Australia around 50,000 y ago, resolving a decades-long controversy. These specimens yielded no endogenous DNA, but protein sequences were able to identify that the eggshell fragments belonged to the extinct, giant flightless bird Genyornis newtoni through the exclusion of other possible bird taxa.…”